Rainer Herken

Skin and hair follicle integrity is crucially dependent on β1 integrin expression on keratinocytes

β1 integrins are ubiquitously expressed receptors that mediate cell–cell and cell–extracellular matrix interactions. To analyze the function of β1 integrin in skin we generated mice with a keratinocyte‐restricted deletion of the β1 integrin gene using the cre–loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of α6β4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin‐5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal–epidermal junction. In contrast, the integrity of the basement membrane surrounding the β1‐deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of β1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiaton and proliferation, and in the formation and/or maintenance of hemidesmosomes.

Bone marrow angiogenesis and mast cell density increase simultaneously with progression of human multiple myeloma

SummaryImmunohistochemical, cytochemical and ultrastructural data showing vivid angiogenesis and numerous mast cells (MCs) in the bone marrow of 24 patients with active multiple myeloma (MM) compared with 34 patients with non-active MM and 22 patients with monoclonal gammopathy of undetermined significance (MGUS) led us to hypothesize that angiogenesis parallels progression of MM, and that MCs participate in its induction via angiogenic factors in their secretory granules.

Aquaporin-4 deficiency in skeletal muscle and brain of dystrophic mdx mice

We report a detailed study of AQP4 expression in the neuromuscular system of mdx mice. Immunocytochemical analysis performed by double immunostaining revealed that mdx mice manifest a progressive reduction in AQP4 at the sarcolemmal level of skeletal muscle fast fibers and that type IIB fibers are the first to manifest this reduction in AQP4 expression. No labeling was observed in the cytoplasm of muscle fibers, indicating that the reduction in sarcolemma staining is not associated with an intracellular compartmentalization of mistargeted protein. By Western blot and RT‐PCR analysis, we found that whereas the total content of AQP4 protein decreased (by 90% in adult mdx mice), mRNA levels for AQP4 remained unchanged. A similar age related reduction in AQP4 expression was found in brain astrocytic end‐feet surrounding capillaries of mdx mice. Morphometric analysis performed after immunogold electron microscopy indicated a reduction of ~85% in gold particles (32±2/μm vs. 4.7±0.61/μm). Western blot experiments conducted using membrane fractions from brain cortex revealed a strong reduction (of 70%) in AQP4 protein in adult mdx mice, and RT‐PCR experiments demonstrated that the reduction was not at transcription level. More interesting was the finding that AQP4 reduction was associated with swelling of astrocytic perivascular processes whose ultrastructural modifications are commonly indicated as an important and early event in the development of brain edema. No apparent reduction in AQP4 was found in mdx stomach and kidney. Our data provide evidence that dystrophin deficiency in mdx mice leads to disturbances in AQP4 assembly in the plasma membrane of fast skeletal muscle fibers and brain astrocytic end‐feet, suggesting that changes in the osmotic equilibrium of the neuromuscular apparatus may be involved in the pathology of muscular dystrophy.—Frigeri, A., Nicchia, G. P., Nico, B., Quondamatteo, F., Herken, R., Roncali, L., Svelto, M. Aquaporin‐4 deficiency in skeletal muscle and brain of dystrophic mdx mice. FASEB J. 15, 90–98 (2001)

Severe Alterations of Endothelial and Glial Cells in the Blood-Brain Barrier of Dystrophic mdx Mice

In this study, we investigated the involvement of the blood‐brain barrier (BBB) in the brain of the dystrophin‐deficient mdx mouse, an experimental model of Duchenne muscular dystrophy (DMD). To this purpose, we used two tight junction markers, the Zonula occludens (ZO‐1) and claudin‐1 proteins, and a glial marker, the aquaporin‐4 (AQP4) protein, whose expression is correlated with BBB differentiation and integrity. Results showed that most of the brain microvessels in mdx mice were lined by altered endothelial cells that showed open tight junctions and were surrounded by swollen glial processes. Moreover, 18% of the perivascular glial endfeet contained electron‐dense cellular debris and were enveloped by degenerating microvessels. Western blot showed a 60% reduction in the ZO‐1 protein content in mdx mice and a similar reduction in AQP4 content compared with the control brain. ZO‐1 immunocytochemistry and claudin‐1 immunofluorescence in mdx mice revealed a diffuse staining of microvessels as compared with the control ones, which displayed a banded staining pattern. ZO‐1 immunogold electron microscopy showed unlabeled tight junctions and the presence of gold particles scattered in the endothelial cytoplasm in the mdx mice, whereas ZO‐1 gold particles were exclusively located at the endothelial tight junctions in the controls. Dual immunofluorescence staining of α‐actin and ZO‐1 revealed colocalization of these proteins. As in ZO‐1 staining, the pattern of immunolabeling with anti–α‐actin antibody was diffuse in the mdx vessels and pointed or banded in the controls. α‐actin immunogold electron microscopy showed gold particles in the cytoplasms of endothelial cells and pericytes in the mdx mice, whereas α‐actin gold particles were revealed on the endothelial tight junctions and the cytoskeletal microfilaments of pericytes in the controls. Perivascular glial processes of the mdx mice appeared faintly stained by anti‐AQP4 antibody, while in the controls a strong AQP4 labeling of glial processes was detected at light and electron microscope level. The vascular permeability of the mdx brain microvessels was investigated by means of the horseradish peroxidase (HRP). After HRP injection, extensive perivascular areas of marker escape were observed in mdx mice, whereas HRP was exclusively intravascularly localized in the controls. Inflammatory cells, CD4‐, CD8‐, CD20‐, and CD68‐positive cells, were not revealed in the perivascular stroma of the mdx brain. These findings indicate that dystrophin deficiency in the mdx brain leads to severe injury of the endothelial and glial cells with disturbance in α‐actin cytoskeleton, ZO‐1, claudin‐1, and AQP4 assembly, as well as BBB breakdown. The BBB alterations suggest that changes in vascular permeability are involved in the pathogenesis of the neurological dysfunction associated with DMD. GLIA 42:235–251, 2003.

Fibrillin-1 and fibrillin-2 in human embryonic and early fetal development

The extracellular glycoproteins fibrillin-1 and fibrillin-2 are major components of connective tissue microfibrils. Mutations in the fibrillin-1 and fibrillin-2 genes are responsible for the phenotypical manifestations of Marfan syndrome and congenital contractural arachnodactyly respectively, which emphasizes their essential roles in developmental processes of various tissues. Consistent with this last notion, organ culture experiments have indirectly suggested morphogenic roles for fibrillins in lung and kidney development. In order to contribute to the understanding of the roles of fibrillins in developmental and morphogenetic events, we have investigated the distribution of fibrillin-1 and fibrillin-2 in human embryonic and early fetal tissues between the 5th and the 12th gestational week, i.e. at the beginning of organogenesis. Fibrillin-1 and fibrillin-2 were localized immunohistochemically using specific monoclonal antibodies, mAb 69 and mAb 48, respectively. Both fibrillins are widely distributed in various human anlagen, from early developmental stages. In most embryonic and early fetal human organs such as skin, lung, heart, aorta, central nervous system anlage, nerves, and ganglia, fibrillin-1 and fibrillin-2 follow the same temporo-spatial pattern of distribution. However, in other organs such as kidney, liver, rib anlagen, notochord fibrillin-1 and fibrillin-2 are distributed differentially. The present paper is focused on this aspect. These results suggest different roles for fibrillin-1 and -2 in the development of these structures.

The collagen type XVIII endostatin domain is co-localized with perlecan in basement membranes in vivo

The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo.

The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo.

SummaryFibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4–10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvement in the differentiation of heart, skeletal and neuronal structures.

Intermediate filament typing of the human embryonic and fetal notochord.

In order to characterize human notochordal tissue we investigated notochords from 32 human embryos and fetuses ranging between the 5th and 13th gestational week, using immunohistochemistry to detect intermediate filament proteins cytokeratin, vimentin and desmin, the cytokeratin subtypes 7, 8, 18, 19 and 20, epithelial membrane antigen (EMA), and adhesion molecules pan-cadherin and E-cadherin. Strong immunoreactions could be demonstrated for pan-cytokeratin, but not for desmin or EMA. Staining for pan-cadherin and weak staining for E-cadherin was found on cell membranes of notochordal cells. Also it was demonstrated that notochordal cells of all developmental stages contain the cytokeratins 8, 18 and19, but not 7 or 20. Some cells in the embryonic notochord also contained some vimentin. Vimentin reactivity increased between the 8th and 13th gestational week parallel to morphological changes leading from an epithelial phenotype to the chorda reticulum which represents a mesenchymal tissue within the intervertebral disc anlagen. This coexpression reflects the epithelial-mesenchymal transformation of the notochord, which also loses E-cadherin expression during later stages. Our findings cannot elucidate a histogenetic germ layer origin of the human notochord but demonstrate its epithelial character. Thus, morphogenetic inductive processes between the human notochord and its surrounding vertebral column anlagen can be classified as epithelial-mesenchymal interactions.

Light and electron microscopical immunohistochemical localization of the small proteoglycan core proteins decorin and biglycan in human knee joint cartilage.

SummaryThe distribution of decorin and biglycan was investigated at the light and electron microscopical level in adult human articular cartilage. In general, the amount of decorin and biglycan was found to decrease with the depth of the layer of the cartilage. Decorin was found in the interterritorial matrix where most of the collagen is located. This fits in well with the assumption that decorin may modulate collagen metabolism. Biglycan was found next to the chondrocytes in the pericellular matrix and is assumed to be responsible for cellular activities. At the ultrastructural level, decorin was localized in the interterritorial matrix and in vesicles in chondrocytes. Biglycan was found, usually though not exclusively in the pericellular matrix. Both small proteoglycans were detected close to and on the collagen fibres and also associated with the more globular structures of the matrix between the fibrils. A double-staining approach revealed that the two molecules could be located along the same collagen fibril. However, staining for biglycan and decorin was not observed simultaneously within the same region of the fibre.

Developmental expression of ZO-1 antigen in the mouse blood-brain barrier.

Tight junction biogenesis during blood-brain barrier development (BBB) in mesencephalon microvessels of mouse embryos of day 9, foetuses of day 15 and 19 and new-born (2-day-old) mice was examined by light and electron microscopy, using monoclonal antibodies recognizing the tight junction peripheral membrane protein ZO-1. A faint spot-like staining began to be recognizable under the light microscope in day 15 vessels in which the endothelial cells showed isolated fusion points between the external plasma membrane leaflets under the electron microscope. A stronger labelling was present in microvessels of day 19 foetuses and new-born animals when the endothelial tight junction appeared completely differentiated. In the immunogold study, gold particles were seen scattered throughout the cytoplasm of endothelial cells of day 15 foetuses. In day 19 foetuses and in the new-born mice, gold particles were located only at the cytoplasmic surfaces of the tight junctions. The results indicate that the ZO-1 protein is a specific molecular marker in the developing brain endothelial tight junctions and that its expression takes place parallel to BBB morphofunctional maturation.